Cystic fibrosis is a human genetic disease of epithelia. Although the survival rate of those suffering with cystic fibrosis has improved in recent years, the median age for patient survival is still only about twenty five to thirty years despite intensive supportive and prophylactic treatment. Today cystic fibrosis remains the most common congenital disease among Caucasians, where it has a prevalence of about one in two thousand live births, and is uniformly fatal. Nearly all patients suffering from the disease develop chronic progressive disease of the respiratory system, the most common cause of death being pulmonary disease. Also, in the majority of cases, pancreatic dysfunction occurs; hepatobiliary and genitourinary diseases are also frequent. Because of the multi-system clinical manifestations of the disease, current methods of treatment for the disease have focused on therapeutic approaches to reduce the symptoms of cystic fibrosis.
For example, U.S. Pat. No. 5,100,647 to Agus, et al, discloses a method for treating cystic fibrosis by administration of the compound sparteine (dodecahydro-7, 14methano-2H, 6H-di-pyrido 1,2-a: 1',2'-e! 1,5! diazocine), acting as a direct exogenous activator of chloride conductants in epithelial airways. U.S. Pat. No. 5,179,001 to Young, et al, discloses a method of treating pulmonary complications associated with cystic fibrosis caused by the gram negative bacterium Pseudomonas aeruginosa. U.S. Pat. No. 4,826,679 to Roy relates to an oral composition for alleviating digestive manifestation in persons afflicted with cystic fibrosis comprising a therapeutic amount of taurine.
Despite much advancement in the treatment of the symptoms of cystic fibrosis very little has been accomplished to effectively "cure" the disease at a molecular level.
One method of gene therapy proposed is U.S. Pat. No. 5,149,797 disclosing a method of site specific alteration of RNA and production of encoded polypeptides. This invention is drawn to correcting the abnormal mRNA present in individual cells, cleaving the mRNA by site directed RNAase followed by introduction of the appropriate oligoribonucleotide followed by endogenous RNA ligase and thus production of a wild-type mRNA encoding a normal protein product which then may be translated to produce the correct protein.
The past few years have brought dramatic advances in our knowledge of the molecular and cellular basis of CF (for reviews see: Collins, F. S., (1992), "Cystic Fibrosis: Molecular Biology and Therapeutic Implications", Science 256, 774-779; Riordan, J. R., (1993), "The Cystic Fibrosis Transmembrane Conductance Regulator", Annu. Rev. Physiol. 55, 609-630; Welsh, M. J., (1995), "Cystic Fibrosis", In the Metabolic and Molecular Basis of Inherited Disease, C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, eds. (New York: McGraw-Hill, Inc.), pp. 3799-3876). We now know that the disease is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation-regulated Cl.sup.- channel located in the apical membrane of involved epithelia. Also, much has been discovered about how CF-associated mutations disrupt protein function, thereby disrupting Cl.sup.- transport across CF epithelia.
Despite these advances, the pathogenesis of CF lung disease, the major cause of morbidity and mortality, is still not understood. Lung disease is characterized by bacterial colonization and chronic airway infection. Many organisms can be involved, but Pseudomonas aeruginosa and Staphylococcus aureus are particularly prominent (Konstan, M. W., (1993), "Infection and Inflammation of the Lung in Cystic Fibrosis", In Cystic Fibrosis, P. B. Davis, ed. (New York: Marcel Dekker, Inc.), pp. 219-276). Chronic bacterial infections progressively destroy the lung, and may ultimately lead to respiratory failure. Several hypotheses have been proposed to explain the pathogenesis of CF lung disease (Davis, P. B., (1993), "Pathophysiology of the Lung Disease in Cystic Fibrosis", In Cystic Fibrosis, P. B. Davis, ed. (New York: Marcel Dekker, Inc.), pp. 193-218; Wine, J. M., (1995), "How do CFTR Mutations Cause Cystic Fibrosis", Curr. Biol. 5, 1357-1359; Pilewski, J. M., (1995), "How do Cystic Fibrosis Transmembrane Conductance Regulator Mutations Produce Lung Disease?", Curr. Opin. Pulm. Med. 1, 435-443; Welsh, M. J., (1995), "Cystic Fibrosis", In the Metabolic and Molecular Basis of Inherited Disease, C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, eds. (New York: McGraw-Hill, Inc.), pp. 3799-3876). However, it has been difficult to relate the characteristic disease abnormality, bacterial colonization and infection of airways, to the characteristic physiologic abnormality, defective transepithelial Cl.sup.- transport.
In other organs affected by CF, disease pathogenesis does not involve bacterial infections. For the sweat glands, pancreas, intestine, and male genital tract, plausible explanations of pathogenesis are based on defective transepithelial Cl.sup.- transport (Quinton, P. M., (1990), "Cystic Fibrosis: A Disease in Electrolyte Transport", FASEB J. 4, 2709-2717.; Welsh, M. J., (1995), "Cystic Fibrosis", In the Metabolic and Molecular Basis of Inherited Disease, C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, eds. (New York: McGraw-Hill, Inc.), pp. 3799-3876). Likewise, as suggested by Quinton, P. M., (1984), "Exocrine Glands", In Cystic Fibrosis, L. M. Taussig, ed. (New York: Thieme-Statton Inc.), pp. 338-375 over a decade ago, defective transepithelial electrolyte transport might somehow be responsible for the pathogenesis of airway infections. In airway epithelia, the loss of CFTR Cl.sup.- channel function, perhaps combined with a secondary defect in Na.sup.+ transport, leads to abnormal transepithelial salt and fluid transport (Boucher, R. C., (1983), "Epithelial Dysfunction in Cystic Fibrosis Lung Disease", Lung 161, 1-17; Jiang, C., (1993), "Altered Fluid Transport Across Airway Epithelium in Cystic Fibrosis", Science 262, 424-427; Smith, J. J. (1993), "Fluid and Electrolyte Transport by Cultured Human Airway Epithelia", J. Clin. Invest. 91, 1590-1597; Smith, J. M. (1994), "Defective Fluid Transport by Cystic Fibrosis Airway Epithelia", J. Clin. Invest. 93, 1307-1311). As a result, the composition of airway surface fluid is abnormal. Joris, L., (1993), "Elemental Composition of Human Airway Surface Fluid in Healthy and Diseased Airways", Am. Rev. Respir. Dis. 148, 1633-1637 and Gilljam, H., (1989), "Increased Bronchial Chloride Concentration in Cystic Fibrosis", Scand. J. Clin. Lab. Invest. 49, 121-124 have shown that airway surface fluid from patients with CF has increased concentrations of Cl.sup.- and Na.sup.+ when compared to that of normal subjects. Until now, however, no one has been able to determine how these abnormal concentrations of ions correlates to increased airway infections in CF patients.
Applicants have now discovered that airway surface fluid in both normal and CF airway surfaces contains a defensin-like factor with broad-spectrum anti-microbial activity. The alteration of the ionic composition of airway surface fluid in CF patients inhibits the activity of this bactericidal factor which explains the increased frequency of airway infections in these patients. The discovery of this link between the abnormal ionic concentrations of airway surface fluid and increased infections provides valuable insight into local pulmonary defense mechanisms and for the prevention and treatment of airway infections in CF patients and in other disease states which are characterized by opportunistic respiratory infections.
It is therefore a primary objective of the present invention to provide a broad spectrum antimicrobial factor isolated from human mucosal epithelial cells.
It is a further objective of the present invention to provide a method of treating chronic airway infections characterized by defective ion transport.
It is another objective of the present invention to provide a method of purifying a broad spectrum antimicrobial factor isolated from human mucosal epithelial cells.
It is yet a further objective of the present invention to provide a therapeutic composition for treating pulmonary infections associated with CF by reducing the salt concentration of the airways in CF patients.
It is yet a further objective of the present invention to provide a method of preparing an antimicrobial composition containing a broad spectrum antimicrobial factor isolated from mucosal epithelial cells.
It is another objective of the present invention to provide a method of treating respiratory bacterial infections which are characterized by abnormal ion concentrations in airway surface fluid.
It is another objective of the present invention to provide a method of treating non-respiratory mucosal infections using a bactericidal factor isolated from mucosal epithelial cells.
It is yet another objective of the present invention to provide a method of testing for the presence of a broad spectrum antimicrobial factor using a sample from a biological source suspected of containing the antimicrobial factor.
Other objects of the invention will become apparent from the description of the invention which follows.